Method for treating atherosclerosis hyperlipidema, lipid deposition on arterial walls, or raising the ratio of high density lipoprotein cholesterol to total cholesterol in serum

ABSTRACT

3,5-Dimethyl-4,6-diphenyl-tetrahydro-2H-1,3,5-thiadiazine-2-thione having the formula: ##STR1## as an antiatherosclerotic agent.

The present invention relates to an antiatherosclerotic agent. Moreparticularly, the present invention relates to3,5-dimethyl-4,6-diphenyl-tetrahydro-2H-1,3,5-thiadiazine-2-thionehaving the formula: ##STR2## useful as an active antiatheroscleroticagent.

Hyperlipidemia (hyperlipemia) is regarded as a major risk factor for theatherosclerosis. Heretofore, a number of antihyperlipidemic agents havebeen studied. Therapeutic agents in this field are likely to be used foran extended period of time in view of the nature of the diseases, andthey are required to be highly safe. However, with respect to nicotinicacid and its derivatives, or clofibrate and its derivatives, which havebeen widely used as antihyperlipidemic agents, various subsidiary illeffects have been reported, and they can hardly be accepted assatisfactory therapeutic agents. For instance, with respect to nicotinicacid and its derivatives, it has been reported that they will bringabout e.g. flashing or gastroenteric troubles. With respect toclofibrate and its derivatives, it is known that they will bring aboute.g. myalgia or hepatic insufficiency, and they are likely to lead togallstone formation. Further, it has been reported that clofibratebrings about hepatic carcinoma on animal experiments. [D. J. Svoboda andD. L. Azarnoff, Cancer Res., 39, 3419 (1979)]

In addition to the question of the safety, there has been a progress inthe study of the pharmacological activities. Reflecting the progress inthe recent years in the study of the lipid metabolism, particularly inthe study of the functional mechanism of serum lipoprotein as atransporter of serum lipid, an attention about the effect of the drughas been drawn not only to the activity of the drug to reduce the lipidconcentration in serum but also to the effect to the lipoprotein. Serumcholesterol constitutes the lipoprotein together with triglyceride,phospholipid and apoprotein. This lipoprotein is generally classifiedinto Cyromicron, VLDL (very low density lipoprotein), LDL (low densitylipoprotein) and HDL (high density lipoprotein) depending upon thedifference in the specific gravity. Among these, Cyromicron, VLDL andLDL are believed to be the lipoproteins which induce atherosclerosis.Whereas, HDL is believed to have functions to transport cholesterol fromperipheral blood vessels to a liver, to form a cholesterol ester or tocontribute to the catabolism of triglyceride, and thus serves for theprevention and regression of the atherosclerosis. Accordingly, for anantihyperlipidemic agent to be developed, it is desired that such anagent has not only the function to reduce the total value of serumcholesterol, but also the functions to reduce LDL-cholesterol and toincrease HDL-cholesterol. A therapeutic agent having both of suchfunctions, exhibits an effect to prevent lipid deposition on arterialwalls, and thus serves as an antiatherosclerotic agent.

The synthesis of 3,5-dimethyl-4,6-diphenyl-2H-1,3,5-thiadiazine-2-thioneuseful as a therapeutic agent of the present invention, is disclosed inChem. Ber., 100, 1602 (1967) and Zeitschrift fuer Chemie, 14, 270(1974). However, no application of this compound to medical orpharmaceutical agents has been known. Further, various derivativeshaving a basic structure of tetrahydro-2H-1,3,5-thiadiazine-2-thionehave been synthesized and have been known as being useful as fungicidesor animal food additives (nutrients). However, nothing has been knownwith respect to their antihyperlipidemic effect and antiatheroscleroticeffect.

As a result of extensive researches for compounds havingantiatherosclerotic effects, the present inventors have found that theabove-mentioned3,5-dimethyl-4,6-diphenyl-tetrahydro-2H-1,3,5-thiadiazine-2-thione(hereinafter referred to simply as "the compound of the formula I") hasexcellent antihyperlipidemic effect and is particularly effective notonly to reduce LDL-cholesterol and increase HDL-cholesterol, but also toprevent the lipid deposition on arterial walls. On the basis of thisdiscovery, it has been found that the compound of the formula I isuseful as an active ingredient for an antiatherosclerotic agent. Theanti-atherosclerotic agent is effective as a preventive and curativepharmaceutical composition for ischemic circulatory organs diseases suchas myocardial infarction, heart attack, cerebral infarction,hypertension or thrombus. Further, it has been found that the compoundof the formula I is a highly safe compound which does not bring aboutside effects to liver, such as hepatomegaly or hepatic insufficiency.

The pharmaceutical composition of the present invention comprises aneffective amount of the compound of the formula I and a pharmaceuticallyacceptable carrier. The effective amount is usually at least 5% byweight, based on the total composition. As the pharmaceuticallyacceptable carrier, there may be mentioned a pharmaceutically acceptablebinder such as a syrup, gum arabic, gelatin, sorbitol, tragacanth gum orpolyvinylpyrrolidone (molecular weight of e.g. about 25,000); anexcipient such as lactose, sugar, corn starch, calcium phosphate,sorbitol or glycine; a lubricant such as magnesium stearate, talc,polyethylene glycol or silica; or a disintegrator such as potato starch.By properly selecting the carrier, the pharmaceutical composition of thepresent invention may be formulated into powders, granules, tablets orcapsules. It is preferably administered orally. However, the manner ofadministration is not restricted to oral administration, and non-oraladministration may be employed.

The daily dose of the compound of the formula I is from 0.01 to 3.0 g,preferably from 0.1 to 1.5 g, for an adult. It is administered from onceto three times per day. The dose may of course be varied depending uponthe age, the weight or the condition of illness of the patient. Now, theusefulness of the compound of the formula I as an antiatheroscleroticagent will be shown by tests. In the following tests, the fractionationof lipoproteins was conducted by a dextran sulfate-MgCl₂ precipitationmethod.

Cholesterol in serum was measured by means of a cholesterol measuringkit (Cholesterol C-Test Wako, manufactured by Wako Junyaku Co., Ltd.),and cholesterol in HDL was measured by means of NC Hi-Set, manufacturedby Nippon Chemiphar Co., Ltd. The quantitative analysis of triglyceridewas conducted by means of a triglyceride measuring kit (TriglycerideC-II-Test Wako, manufactured by Wako Junyaku Co., Ltd.).

In the following Tables, cholesterol is referred to as "Chol", andtriglyceride is referred to as "TG".

Further, the change rate in the following Tables, was calculated by thefollowing equation.

    Change rate(%)=(A-B/A)×100

where A is the amount of serum lipids (mg/dl) of the control group, andB is the amount of serum lipids (mg/dl) of the group to which thetherapeutic agent was administered.

TEST 1 The antihyperlipidemic activity in emulsion-inducedhyperlipidemic rats

Male S.D. rats weighing 80-90 g (4 weeks old) were used. They weredivided into groups of 5 to 6 rats each. The test compounds suspended in0.5% CMC-Na(carboxymethyl cellulose sodium salt) were given to the ratsin a daily dose of 4 ml/kg via stomach tube every 10:00 a.m. After 30min., lipids emulsion having the following composition was orally givento the rats in an amount of 2.5 ml per rat. During the experimentalperiod of 3 days, the rats were fed on a standard commercial diet andwater ad libitum. At the end of the period, the rats were fasted for 16hours and then blood samples were obtained from inferior vena cava. Thetotal cholesterol and triglyceride, and HDL cholesterol were measured.The results are shown in Table 1.

    ______________________________________                                        Composition of emulsion:                                                      ______________________________________                                        Cholesterol              22.5   g                                             Cholic acid sodium salt  10.0   g                                             Sucrose                  90.0   g                                             Olive oil                150.0  g                                             Water                    ×                                                                              ml                                            Final volume             300.0  ml                                            ______________________________________                                    

                  TABLE 1                                                         ______________________________________                                                    Serum lipid                                                       Test     Dose     Total Chol                                                                              HDL-Chol TG                                       compounds                                                                              mg/kg    mg/dl     mg/dl    mg/dl                                    ______________________________________                                        Compound of                                                                            300      145       22.1     97.4                                     formula I         (-45.0) *.sup.2                                                                         (+80.6)  (-36)                                    Clofibrate                                                                             300       96.4     16.8     87.7                                     *.sup.1           (-63.5)   (+37.0)  (-42.7)                                  Control  --       266.8     12.3     153.0                                    ______________________________________                                         ##STR3##                                                                     - -                                                                            *.sup.2 Change rate (%)                                                  

TEST 2 The antihyperlipidemic activity in Triton-induced hyperlipidemicrats

Male S.D. rats weighing 230-250 g (7 weeks old) were divided into groupsof 5 rats each and injected with a physiological saline solution ofTriton Wr-1339 in a dose of 250 mg/kg (5 ml/kg) i.v. through the tailvein. After 24 hours (method A) or 43 hours (method B), blood sampleswere taken from the inferior vena cava with the animals. The testcompounds suspended in 0.5% CMC-Na were given to the rats in an amountof 2 ml/kg via stomach tube 1 hour before and 5 hours after (in the caseof method A) or 1 hour before and 23 hours after (in the case of methodB) the injection of Triton solution. Control rats received the samevolume of the vehicle. The animals were fasted during the experimentalperiod. The total cholesterol and triglyceride in the serum weremeasured. The results are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                                          Change rate (%)                                                                            Change rate (%)                                Test      Dose    after 24 hours                                                                             after 43 hours                                 compounds mg/kg   Chol     TG    Chol   TG                                    ______________________________________                                        Compound of                                                                             150     -6.0     +21.8 -43.5  -65.7                                 the formula I                                                                 Clofibrate *.sup.1                                                                      300     -5.0     +89.7  -0.5  +150.1                                Clinofibrate *.sup.2                                                                    150     -21.0    -33.0 -38.0  -52.0                                 ______________________________________                                         *.sup.1 As identified in Table 1.  -                                          ##STR4##                                                                 

As is evident from Table 2, the compound of the formula I showed adistinc lipid reducing activity in the case of method B although nolipid reducing activity was observed in the case of method A.

TEST 3 The antihyperlipidemic activity in cholesterol-fed hyperlipidemicrats

Male S.D. rats weighing 80-90 g (30 days old) were divided into groupsof 5 rats each. The test compounds suspended in 0.5% CMC-Na were givento the rats in a daily dose of 0.4 ml per 100 g rat via stomach tubeevery 10:00 a.m. for 7 or 14 days. Control groups were given an equalvolume of the vehicle. During the experimental period, the animals werefed on a 1% cholesterol diet (powdered form) prepared by the method ofTensho et al. [Yakugaku Zasshi, 92, 878 (1972)]. At the end of theperiod, the animals were fasted for 16 hours and then blood samples wereobtained from inferior vena cava. The total cholesterol, triglycerideand HDL cholesterol in serum were measured. After sacrificed, the liverswere excised, washed with physiological saline, blotted on filter paperand weighed. The degree of hepatomegaly was calculated by the followingequation.

    Degree of hepatomegaly (%)=(D-C/C)×100

where C is the liver weight (g) per 100 g body weight in the controlanimal, and D is the liver weight (g) per 100 g body weight in the drugtreated animal.

The results on the 7th day are shown in Table 3, and the results on the14th day are shown in Table 4.

                  TABLE 3                                                         ______________________________________                                        7th day                                                                                                    Degree                                                     Serum lipid        of                                                               Total-                   hepato-                              Test    Dose    Chol      HDL-Chol                                                                              TG     megaly                               compounds                                                                             mg/kg   mg/dl     mg/dl   mg/dl  (%)                                  ______________________________________                                        Compound                                                                              150     385       17.3     66.2  +12.0                                (I)             .sup. (-11.7)*.sup.2                                                                    (-4.4)  (-46.1)                                             100      294.7    19.2     92.7  ±0                                                (-32.4)   (+6.1)  (-24.5)                                              50      289.8    18.1    104.0  +2.0                                                 (-33.6)   (0)     (-15.3)                                              10      421.7    15.8    105.9  +4.0                                                  (-3.3)   (-12.7) (-13.8)                                     Clofibrate*.sup.1                                                                     150      332.2    11.8    111.8  +20.0                                                (-23.8)   (-34.8) (-9.0)                                               50      417.5    14.4    133.5  +6.0                                                  (-4.3)   (-20.4) (+8.7)                                      Control --       426.2    18.1    112.8  --                                   ______________________________________                                         *.sup.1 As identified in Table 1.                                             *.sup.2 Change rate (%)                                                  

                  TABLE 4                                                         ______________________________________                                        14th day                                                                               Serum lipid                                                          Test           Total-    HDL-          Degree of                              com-   Dose    Chol      Chol   TG     hepato-                                pounds mg/kg   mg/dl     mg/dl  mg/dl  megaly (%)                             ______________________________________                                        Com-   150      228.6      21.5  77.1  +5.3                                   pound (I)      .sup. (-60.9)*.sup.2                                                                    (+147.1)                                                                             (-53.8)                                              100      271.3      24.9  92.2  +10.5                                                 (-53.7)   (+186.2)                                                                             (-44.8)                                               50      389.2      20.5  87.7  +1.8                                                  (-33.5)   (+135.6)                                                                             (-47.5)                                               10      336.5      20.0  92.5  +5.3                                                  (-42.5)   (+129.9)                                                                             (-44.6)                                       Clofi- 150      420.0      15.8  117.7 +22.8                                  brate*.sup.1   (-28.3)    (+68.1)                                                                             (-29.8)                                               50      418.4      9.4   116.6 +8.8                                                  (-28.5)    (+8.0)                                                                              (-30.1)                                       Control                                                                              --       585.4      8.7   166.9 --                                     ______________________________________                                         *.sup.1 As identified in Table 1                                              *.sup.2 Change rate (%)                                                  

The compound of the formula I showed a strong lipid reducing activityand HDL-cholesterol increasing activity on the 14th day although suchactivities are not distinct very much on the 7th day. Further, it wasfound to have no hepatomegaly.

TEST 4 The effect of the compound of the formula I on experimentalatherosclerosis in rabbits induced by cholesterol diet

Twenty-five male rabbits weighing 1.8-2.0 kg were used. They were fedwith basic chow (Nippon clea, CR-3) for a stabilizing period of 5 days.After this period, they were fed with the chow containing 1%cholesterol, 100 g per head a day for a week (7 days) and plasmacholesterol was measured using a blood sample collected from acuriclevein.

According to the results of cholesterol values measured, 14 animals ofhigh cholesterol plasma level were selected. They were divided into twogroups to have nearly equal means and S.D. values of plasma cholesterol.One group of 7 rabbits was fed with the chow containing 1% cholesteroland another group of 7 rabbits was fed with the chow containing 1%cholesterol and 0.1% compound of the formula I in an amount of 100 g perhead a day for 8 weeks.

Rabbits were sacrificed at the end of 8 weeks. Aortic arch and thoracicaorta were excised, and macroscopical changes were examined using theSudan III stain method. According to the results of gross observation ofthe internal surface stained by Sudan III, the ratios of theatherosclerotic plaque area (the area stained by Sudan III) to the wholeinternal surface area were calculated. The ratio of inhibition ofatherosclerosis of the compound of the formula I was obtained bycomparing the average value with the value of the control.

                  TABLE 5                                                         ______________________________________                                                         Atherosclerotic plaque area                                                   stained by Sudan III (%)                                                      Aortic Thoracic Total                                                         arch   aorta    aorta                                        ______________________________________                                        Control (E)        90.1     34.1     48.0                                     Treated with compound (I) (F)                                                                    77.7     11.0     26.3                                     Ratio of inhibition of                                                                           13.8     67.7     45.3                                     atherosclerosis                                                               (Ratio of Improvement) (G) (%)*                                               ______________________________________                                         ##STR5##                                                                 

It was confirmed that the compound of the formula I inhibitsexperimental atherosclerosis on rabbits raised with 1% cholesterol diet.

TEST 5 The activities against serum lipid, the degree of hepatomegalyand the hepatic drug metabolizing systems of normal rats

Male S.D. rats (6 weeks old) were divided into groups of 5 rats each.The test compounds suspended in 5% gum arabic were given to the ratsonce a day for seven days in a daily dose of 300 mg/kg. Then, the ratswere fasted for one day, blood samples were taken, and the totalcholesterol, triglyceride, HDL-cholesterol in the serum werequantitatively analyzed. Further, the liver weight was measured, and thedegree of hepatomegaly was determined by comparing the weight ratio per100 g of the body weight with that of the control group. Furthermore,GOT and GPT values were measured, and the activities of cytochrome P-450and glutathione related enzymes were also measured. These values werecompared with those of the control groups to investigate anyabnormality. The results are shown in Table 6.

                  TABLE 6                                                         ______________________________________                                                 Serum lipid                                                          Test           Total-    HDL-          Degree of                              com-   Dose    Chol      Chol   TG     hepato-                                pounds mg/kg   mg/dl     mg/dl  mg/dl  megaly (%)                             ______________________________________                                        Com-   300     39.2       29.2   37.5   +7.0                                  pound (I)      .sup. (-16.2)*.sup.2                                                                     (-1.0)                                                                              (-21.0)                                       Clofi- 300     20.6       11.0   39.2  +72.0                                  brate*.sup.1   (-56.0)   (-62.7)                                                                              (-17.5)                                       Control                                                                              --      46.8       29.5   47.5  --                                     ______________________________________                                         *.sup.1 As identified in Table 1.                                             *.sup.2 Change rate (%)                                                  

It is evident from Table 6 that the compound of the formula I bringsabout no substantial hepatomegaly in the normal rats, and it ramarkablyreduces LDL-cholesterol. No hepatic insufficiency was observed from theGOT and GPT values. Further, no abnormalities were observed with respectto the activity against cytochrome P-450 or other liver microsome drugmetabolizing systems, and against glutathione related enzymes.

TEST 6 Mutagenicity test

The mutagenicity was tested by using Escherichia coli (WP2UVrA) andSalmonella (TA98, TA100, TA1535 and TA1538) in the absence or presenceof a drug metabolizing enzymes S-9 mix of rat liver. The compound of theformula I did not show any mutagenicity up to a concentration as high as5000 μg/Plate, thus indicating that it is a highly safe compound.

TEST 7 Acute toxicity test

The test compounds suspended in 0.5% CMC-Na were administered p.o. orinjected i.p. to male ddY mice. The acute toxicity was dertermined basedon the mortality after seven days. The compound of the formula I showedno toxicity up to the dose of 8,000 mg/kg in the case of the oraladministration and up to the dose of 4,000 mg/kg in the case of theabdominal administration, thus indicating that it is a safe compoundhaving a low acute toxicity.

Now, examples will be given for various formulations containing thecompound of the formula I.

Tablets

    ______________________________________                                        Composition (4,000 tablets)                                                   ______________________________________                                        Compound of the formula (I)                                                                           500    (g)                                            Potato starch           334                                                   Carboxymethyl cellulose 87.5                                                  Polyvinyl alcohol       61                                                    Magnesium stearate      17.5                                                                          1,000                                                 ______________________________________                                    

The above ingredients in the respective amounts were introduced into atwin shell mixer and uniformly mixed. This power mixture was tableted bya direct compression method to obtain tablets having a weight of 250 mgper tablet.

Capsules

    ______________________________________                                        Composition (10,000 capsules)                                                 ______________________________________                                        Compound of the formula I                                                                             2,500  (g)                                            Potato starch           400                                                   Magnesium stearate      100                                                                           3,000                                                 ______________________________________                                    

The above ingredients in the respective amounts were introduced into atwin shell mixer and uniformly mixed. This powder mixture was packed inhard gelatin capsules in an amount of 300 mg per capsule.

Powder

    ______________________________________                                        Composition:                                                                  ______________________________________                                        Compound of the formula I                                                                             200    (g)                                            Lactose                 800                                                                           1,000                                                 ______________________________________                                    

The above ingredients in the respective amounts were introduced into atwin shell mixer and uniformly mixed to obtain a powder.

We claim:
 1. A method of reducing the incidence or severity ofatherosclerosis in a mammal comprising administering to said mammal anamount effective to reduce the incidence or severity of atherosclerosisof a pharmaceutical composition comprising3,5-dimethyl-4,6-diphenyl-tetrahydro-2H-1,3,5-thiadiazine-2-thionehaving the formula: ##STR6## and a pharmaceutically acceptable carrier.2. A method of treating hyperlipidemia in a mammal in need of suchtreatment comprising administering to said mammal anantihyperlipidemically effective amount of a pharmaceutical compositioncomprising3,5-dimethyl-4,6-diphenyl-tetrahydro-2H-1,3,5-thiadiazine-2-thionehaving the formula: ##STR7## and a pharmaceutically acceptable carrier.3. A method of reducing lipid deposition on the arterial walls of amammal comprising administering to said mammal an amount effective toreduce the lipid deposition on the arterial walls of a pharmaceuticalcomposition comprising3,5-dimethyl-4,6-diphenyl-tetrahydro-2H-1,3,5-thiadiazine-2-thionehaving the formula: ##STR8## and a pharmaceutically acceptable carrier.4. A method of raising the ratio of high density lipoprotein cholesterolin serum to total cholesterol in serum in a mammal in need of suchraising of ratio comprising administering to said mammal a ratio raisingeffective amount of a pharmaceutical composition comprising3,5,-dimethyl-4,6-diphenyl-tetrahydro-2H-1,3,5-thiadiazine-2-thionehaving the formula: ##STR9## and a pharmaceutically acceptable carrier.